The function of numerous proteins requires the modification of multiple glutamic acid residues to γ-carboxyglutamate. Among these vitamin K-dependent (VKD) coagulation proteins, factor IX (FIX; Christmas factor), factor VII (FVII), and prothrombin are the best known. The observation that a knock-out of the gene for matrix Gla protein results in calcification of the mouse's arteries (Luo et al. (1997) “Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein” Nature 386:78-81) emphasizes the importance of the vitamin K cycle for proteins with functions other than coagulation. Moreover, Gas6 and other Gla proteins of unknown function are expressed in neural tissue and warfarin exposure in utero results in mental retardation and facial abnormalities. This is consistent with the observation that the expression of VKD carboxylase, the enzyme that accomplishes the Gla modification, is temporally regulated in a tissue-specific manner with high expression in the nervous system during early embryonic stages. Concomitant with carboxylation, reduced vitamin K, a co-substrate of the reaction, is converted to vitamin K epoxide. Because the amount of vitamin K in the human diet is limited, vitamin K epoxide must be converted back to vitamin K by vitamin K epoxide reductase (VKOR) to prevent its depletion.
VKOR is a polytopic membrane protein of the endoplasmic reticulum (ER). It is responsible for the conversion of vitamin K epoxide (KO) to vitamin K and is highly sensitive to inhibition by coumarin drugs, such as warfarin. Warfarin inhibition of VKOR reduces the availability of reduced vitamin K (vitamin K hydroquinone, KH2), which is a cofactor for γ-glutamyl carboxylase that catalyzes the functionally critical post-translational modification of a family of vitamin K-dependent proteins involved in blood coagulation, bone homeostasis, signal transduction, and cell proliferation. Bioinformatic analyses showed that VKOR is a member of a large family of homologues (VKORH) widely distributed throughout evolution. Characterizations of the human VKOR and VKORH from bacteria have yielded a large amount of structure-function information, but some is contradictory. A four-transmembrane domain (TMD) model for human VKOR was proposed based on the crystal structure of VKORH from Synechococcus sp. This model is different from a previous three-TMD topology model in terms of the location of the N-terminus and the conserved cysteine (C43 and C51) loop between the first and second TMD.
The present invention provides methods and compositions for producing vitamin K dependent proteins.